Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Haklai, Roni; Kloog, Yoel

doi:10.1021/bi00388a004

Cited by 9 publications

(12 citation statements)

References 43 publications

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“…PCM was assayed essentially as described [10], using 100/~g gelatin, 10-20/H enzyme preparation, 2.0/~M S-…”

Section: Methodsmentioning

confidence: 99%

“…The gels were then frozen on dry ice and sliced into 2-ram sections each of which was crushed in a small scintillation vial containing 100-200 ,ul of 25 mM sodium phosphate buffer (pH 7.4), 14 mM ~-mercaptoethanol and 0.1070 Nonidet P-40. The vials were shaken for 12 h at 4°C and their PCM activity was then measured in 20-/d aliquots, p-(Chloromercuri)benzoate-agarose chromatography (Bio-Rad Affi Gel-501 was performed as follows: the column (20 ml matrix) was loaded with 100-120 ml of the cytosolic PCM preparation [10] (500-700 mg protein) so as to maintain a ratio of approx. 1 mg protein/0.2/~mol p-(chloromercuri)benzoate.…”

Section: Methodsmentioning

confidence: 99%

“…1 mg protein/0.2/~mol p-(chloromercuri)benzoate. The column was washed with 40 mM sodium phosphate buffer (pH 7.5) containing antiproteases [10] (buffer A) until all non-bound proteins were eluted. Bound proteins were then eluted with 14 mM ~-mercaptoethanol in buffer A.…”

Section: Methodsmentioning

confidence: 99%

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Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells

et al. 1988

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Two distinct protein carboxyl methyltransferases (PCM) were identified in the electric organ of Torpedo ocellata. They were separated from each other in the active form by means of nondenaturing gel electrophoresis and by p-(chloromercufi)benzoate-agarose chromatography, and were individually identified by specific polyclonal antibodies. The existence of at least two distinct PCMs in eucaryotic cells raises the possibility that these enzymes are involved in distinct transmethylation reactions.Protein carboxyl methyltransferase; Protein methylation

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“…PCM was assayed essentially as described [10], using 100/~g gelatin, 10-20/H enzyme preparation, 2.0/~M S-…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells

et al. 1988

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“…These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology.In all vertebrate tissues examined to date, a cytosolic L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) catalyzes the transfer of the methyl group from S-adenosylmethionine to L-isoaspartyl or D-aspartyl residues (3,4,14,25,27). These unusual residues can accumulate in all proteins via the spontaneous decomposition of normal L-aspartyl and L-asparaginyl residues (11, 33) and can inhibit the function of the modified proteins and/or the normal pathways for their enzymatic degradation (12,16,19,22).…”

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confidence: 99%

Distribution of an L-isoaspartyl protein methyltransferase in eubacteria

Clarke

1992

J Bacteriol

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A protein carboxyl methyltransferase (EC 2.1.1.77) that recognizes age-damaged proteins for potential repair or degradation reactions has been found in all vertebrate tissues and cells examined to date. This enzyme catalyzes the transfer of methyl groups from S-adenosylmethionine to the carboxyl groups of D-aspartyl or L-isoaspartyl residues that are formed spontaneously from normal L-aspartyl and L-asparaginyl residues. A similar methyltransferase has been found in two bacterial species, Escherichia coli and Salmonella typhimurium, suggesting that this enzyme performs an essential function in all cells. In this study, we show that this enzyme is present in cytosolic extracts of six additional members of the a and y subdivisions of the purple bacteria: Pseudomonas aeruginosa (,y), Rhodobacter sphaeroides (a), and the 'y enteric species Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratia marcescens. DNA probes from the E. coli methyltransferase gene hybridized only to the chromosomal DNA of the enteric species. Interestingly, no activity was found in the plant pathogen Erwinia chrysanthemi, a member of the enteric family, nor in Rhizobium meliloti or Rhodopseudomonas palustris, two members of the a subdivision. Additionally, we could not detect activity in the four gram-positive species Bacillus subtilis, B. stearothermophilus, Lactobacillus casei, and Streptomyces griseus. The absence of enzyme activity was not due to the presence of inhibitors in the extracts. These results suggest that many cells may not have the enzymatic machinery to recognize abnormal aspartyl residues by methylation reactions. Since the nonenzymatic degradation reactions that generate these residues occur in all cells, other pathways may be present in nature to ensure that these types of altered proteins do not accumulate and interfere with normal cellular physiology.In all vertebrate tissues examined to date, a cytosolic L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77) catalyzes the transfer of the methyl group from S-adenosylmethionine to L-isoaspartyl or D-aspartyl residues (3,4,14,25,27). These unusual residues can accumulate in all proteins via the spontaneous decomposition of normal L-aspartyl and L-asparaginyl residues (11, 33) and can inhibit the function of the modified proteins and/or the normal pathways for their enzymatic degradation (12,16,19,22). The enzymatic methylation of these altered residues in vitro can lead to their conversion to L-aspartyl residues via a nonenzymatic repair pathway (9,16,17,21).A similar methyltransferase, characterized by its ability to methylate L-isoaspartyl-containing peptides, has also been detected in the cytosolic fraction of procaryotic cells such as Salmonella typhimurium (26) and Escherichia coli (8). The enzyme from the latter source has been purified, and the nucleotide sequence and chromosomal location of its structural gene pcm (59 min) have been determined (8). The deduced amino acid sequence of the E. coli enzyme is 31% identical to t...

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“…1992a). plants (Mudgett and Clarke, 1993) and a variety of invertebrates and vertebrates (Barten and O'Dea, 1990;Clarke, 1985;Haklai and Kloog. 1987;O'Connor.…”

Section: Covalent Modification Of Asparaginyl and Aspartyl Residues Amentioning

confidence: 99%

Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins

Visick

Clarke

1995

Molecular Microbiology

112

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SummaryProteins, like DNA, are subject to various forms of damage that can render them non-functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can tie replaced by de novo biosynthesis, cells -especially those subjected to stress or nutrient limitation -have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non-functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been descritied. First, chaperones not only assist in proper de novo folding of proteins but also provide an Important means of restoring activity to conformationally damaged proteins. Second, enzymatic 'repair' systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteotysis provides a 'last-resort' means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods In stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.

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Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Cited by 9 publications

References 43 publications

Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells

Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells

Distribution of an L-isoaspartyl protein methyltransferase in eubacteria

Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins

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